The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to the use of bacteriophages for reducing levels of Escherichia coli O157, and/or treating or preventing Escherichia coli O157 infection or diseases caused thereby, as well as methods for producing said bacteriophages.
Human enteric infections with enterohemorrhagic E. coli O157 are a significant public health problem in many countries. They lead to diarrhea and to serious complications that include hemorrhagic colitis, the hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and death.
Healthy cattle carry E. coli O157 and humans acquire E. coli O157 infections most often by consuming food and water that has been contaminated with faeces from these animals. The demand for effective control of E. coli O157 infections at the pre-harvest level is high because it is generally believed that reducing, or eliminating E. coli O157 infections in cattle and other food animals will dramatically reduce the number of E. coli O157 infections in humans. Benefits occur not only through production of safer food, but through reduced transmission of E. coli O157 among cattle and dissemination in water and the environment. Probiotic bacteria, dietary management, and vaccination with relevant antigens could reduce shedding of E. coli O157 by cattle, however these approaches are poorly developed, or only marginally effective at this time.
Zhao et al. (Journal of Clinical Microbiology 36, 641-7, 1998; U.S. Pat. No. 5,965,128) showed that a mixture of probiotic bacteria, comprised of 18 isolates of E. coli and one Proteus mirabilis strain, reduced shedding of E. coli O157 by cattle. The mechanism by which the probiotic bacteria reduced the shedding of E. coli O157 is not known and the probiotic bacteria are not available commercially.
Hovde et al. (Applied and Environmental Microbiology 65, 3233-5, 1999) showed that higher energy, lower fiber diets reduced the duration of shedding of E. coli O157 by experimentally infected animals.
To date there are no reports of successful vaccination of cattle against carriage of E. coli O157.
Barrow et al. (Barrow et al., 1998, Clin Diag Lab Imm 5:294-298) demonstrated that bacteriophages can control septicemia and meningitis, both non-enteric infections, in a variety of animals. Both of these infections are systemic, rather than intestinal infections leading to diarrheal disease. The bacteriophages were not used to control E. coli O157, or other zoonotic enteric pathogens affecting humans.
Alisky et al. (Alisky et al., 1998, J Infect 36: 5-15) presented this recent review article that provides an historical perspective on the use of bacteriophages for controlling infections and current research in this area, which has been spurred by the emergence of antibiotic-resistant bacterial pathogens.
Bacteriophages have not been used to control E. coli O157 infections in cattle. Kudva et al. (Applied and Environmental Microbiology 65, 3767-73, 1999) recently reported that bacteriophages could kill E. coli O157 in pure culture, and speculated that bacteriophages could be used to control E. coli O157 in animals. However, the ability of phages to kill E. coli O157 in pure culture has been known for some time and forms the basis for a phage typing scheme for E. coli O157. It is of note that this in vitro effect of bacteriophages cannot be extended to therapeutic use in cattle without further research to establish that candidate bacteriophages survive in the bovine gastro-intestinal tract and retain the ability to infect and kill E. coli O157 in vivo. Moreover, bacteriophages for use in vivo must not adversely affect the health of treated animals.
Smith et al. (Smith et al., 1987, J. Gen. Micro. 133: 1111-1126) showed that experimental diarrhea in 6-12 hour old calves due to infection with certain enterotoxigenic strains of E. coli could be controlled by specific bacteriophages infecting those strains of bacteria. As will be appreciated by one knowledgeable of the art, serotypes of E. coli are defined by the presence of a combination of three known antigens on the surface of the cells and are a form of classification of different strains of E. coli. The antigens are O-antigens (somatic carbohydrate component of the cell wall lipopolysaccharide), H-antigens (flagellaxe2x80x94organelles involved with cell locomotion), and K antigens (polysaccharide capsules or pill). The identity of these antigens on individual E. coli is determined by agglutionation tests using specific, highly cross-adsorbed anti-sera. The O157:H7 serotype is one of many different known serotypes of E. coli. The strains used by Williams Smith are of different serotypes than O157:H7 (ie. different O, K, and H antigens). That is, these strains belong to the group of pathogens called enterotoxigenic E. coli, which cause disease by a mechanism distinct from that of E. coli O157. Furthermore, it is of note that the phages used by Williams Smith would not work on E. coli O157:H7 because it lacks the appropriate K antigen receptor. Furthermore, Williams Smith did not show that bacteriophages could be used to eliminate human zoonotic pathogens carried asymptomatically by cattle. It is also of note that the activity of the bacteriophages towards target organisms in host matrices and their stability in host matrices were not evaluated. Finally, the bacteriophages were not used to control E. coli O157, or other zoonotic enteric pathogens affecting humans.
U.S. Pat. Nos. 5,766,892 and 5,688,501 describe methods to treat bacterial infections in animals using bacteriophages that have been altered to resist host defense mechanisms. The bacteriophages were not used to control E. coli O157, or other zoonotic enteric pathogens affecting humans, but rather were serially administered to an animal""s circulatory system and recovered so as to enrich for phage capable of surviving in the host circulatory system. Thus, these patents teach that phage must be modified to avoid the host immune system in order to be effective treatments.
According to a first aspect of the invention, there is provided a pharmaceutical composition comprising at least one bacteriophage selected from the group consisting y of: V4, V5, V5-re-isolated, V7, V8, V11 and V14.
According to a second aspect of the invention, there is provided a method of reducing levels of E. coli O157 in a ruminant animal comprising:
administering to a ruminant animal having E. coli O157 within its gastrointestinal tract at least one bacteriophage selected from the group consisting of: V4, V5, V5-re-isolated, V7, V8, V11, and V14; and
retaining said ruminant animal under conditions such that said bacteriophage infect and lyse said E. coli O157,
thereby reducing levels of E. coli O157 within the gastrointestinal tract of said ruminant animal.
According to a third aspect of the invention, there is provided a method of selecting and isolating bacteriophages capable of lysing E. coli O157 comprising:
administering to a ruminant animal having E. coli O157 within its gastrointestinal tract at least one bacteriophage selected from the group consisting of: V4, V5, V5-re-isolated, V7, V8, V11, and V14;
recovering gastrointestinal content from said ruminant animal;
isolating bacteriophage from said gastrointestinal content;
plating said isolated bacteriophage onto a suitable host; and
purifying the resulting plaques.
According to a fourth aspect of the invention, there is provided a method of reducing levels of E. coli O157 in a matrix comprising:
administering to the matrix containing E. coli O157 therein at least one bacteriophage selected from the group consisting of: V4, V5, re-isolated V5, V7, V8, V11 and V14; and
retaining said matrix under conditions such that said bacteriophage infect and lyse said E. coli O157,
thereby reducing levels of E. coli O157 within the matrix.
According to a fifth aspect of the invention, there is provided a method of reducing the severity of, or preventing a disease caused by E. coli O157 comprising:
administering to an individual in need of such treatment a pharmaceutical composition comprising at least one bacteriophage selected from the group consisting of: V4, V5, V5-re-isolated, V7, V8, V11 and V14; and
retaining said individual under conditions such that said bacteriophage infect and lyse said E. coli O157,
thereby reducing levels of E. coli O157 within the gastrointestinal tract of said individual.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
Definitions
As used herein, E. coli O157 refers to the prototype of the enterohemorrhagic E. coli (EHEC) which cause hemorrhagic colitis and the hemolytic uremic syndrome in humans, and includes EHEC O157:H7 and O157:H-.
As used herein, xe2x80x9cmatrixxe2x80x9d or xe2x80x9cmatricesxe2x80x9d refers to physical environments, for example but by no means limited to, physical environments that approximate the conditions in the gastrointestinal tract, in water or in manure.
As used herein, xe2x80x9cgastrointestinal contentsxe2x80x9d comprises the four stomachs, small intestine and large intestine of a ruminant animal.
As used herein, xe2x80x9ctreatedxe2x80x9d refers to animals administered a pharmaceutical composition whereas xe2x80x9cuntreatedxe2x80x9d refers to control animals.
As used herein, xe2x80x9cruminant animalxe2x80x9d refers to, for example, cattle, sheep, buffalo and goats, which convert plant material to food and fiber.
As used herein, xe2x80x9ceffective amountxe2x80x9d refers to the administration of an amount of a given compound that achieves the desired effect.
As used herein, xe2x80x9cbacteriophages V4, V5, V7, V8, V11 or V14xe2x80x9d refers to bacteriophage phenotypically identical with bacteriophages V4, V5, V7, V8, V11, V14 respectively (Ahmed et al., 1987, Journal of Infectious Diseases 155:806-9). As will be well-known to one knowledgeable in the art, different isolates of a given bacteriophage may vary at the nucleic acid sequence level; however, these bacteriophage are considered to be identical provided they have similar phenotypes. As will be apparent to one knowledgeable in the art, different isolates may be prepared using means known in the art, for example, random chemical or ionizing radiation, recombinant DNA techniques, interbreeding and the like. It is of note that methods for phenotyping bacteriophage include, for example, determining host range.
As used herein, xe2x80x9cbacteriophage V5-re-isolatedxe2x80x9d refers to a bacteriophage derived from V5 following growth in vivo in ruminant animal.
Enteric bacterial pathogens, such as E. coli O157:H7 , are differentially sensitive to the bacteriophages that infect its species. This property has been exploited to develop useful bacteriophage-based schemes for sub-typing enteric bacteria for epidemiological purposes. However, this property makes the development of phage therapy for enteric pathogens difficult because ruminant animals may carry bacteriophage-resistant as well as sensitive strains of the pathogen that are capable of causing disease in humans.
To be used successfully to control human enteric pathogens in ruminant animals, bacteriophages must be capable of killing a broad spectrum of strains within the target species of pathogen and be effective in host matrices. Furthermore, the bacteriophages must survive in the ruminant gastrointestinal tract and retain the ability to infect and kill E. coli O157 in vivo. Moreover, bacteriophages for use in vivo must not adversely affect the health of treated animals. In this regard, rational selection of bacteriophages appropriate for phage therapy can be made based on information about the types of bacteria to be controlled, the reaction of potential bacteriophages with them, the activity of the bacteriophages against target organisms in host matrices, and the stability of the bacteriophages in the host matrices.
The invention involves a novel approach to control E. coli O157 in ruminant animals. It involves oral administration of bacteriophages lytic to E. coli O157 to ruminant animals to reduce or eliminate E. coli O157 in their gastrointestinal tract. It also involves a process for selecting lytic bacteriophages suitable for this purpose.
In other embodiments, a pharmaceutical composition comprising at least one of the bacteriophages is prepared. As will be appreciated by one knowledgeable in the art, the pharmaceutical composition may be used to treat or prevent E. coli O157 infections in mammals in need of such treatment, for example, humans. In addition, at least one bacteriophage may be used to reduce levels of or eliminate E. coli O157 in matrices, for example, well water, municipal water, ground water, manure, foods, produce, and the like.
In one embodiment of the invention, there is provided a method for reducing levels of E. coli O157 in ruminant animals by oral administration of a pharmaceutical composition comprising at least one bacteriophage selected from the group consisting of bacteriophages V4, V5, V7, V8, V11, V14 (Ahmed et al., 1987, Journal of Infectious Diseases 155:806-9) and combinations thereof to ruminant animals. These bacteriophage have previously been used in phage-typing to identify strains of E. coli O157 in vitro but have not been used to reduce levels of E. coli O157 in ruminant animals. As discussed above, the ability of bacteriophage to kill E. coli strains in vitro does not guarantee their effectiveness in vivo.
For use, the pharmaceutical composition comprising at least one of bacteriophages V4, V5, V5-re-isolated, V7, V8, V11 or V14 is prepared as described below. The pharmaceutical composition is then administered to a bovine animal having E. coli O157 within its gastrointestinal tract. As discussed below, administration of the bacteriophage(s) results in decreased levels of fecal shedding immediately following challenge (p less than 0.05) and eliminates the organism from treated but not untreated cattle after 8 days following challenge. Thus, the pharmaceutical composition enters the digestive tract of the cow and lyses the E. coli O157, thereby reducing levels of the organism within the host and eliminating shedding of the bacteria by the host. As will be apparent to one knowledgeable in the art, elimination of the bacteria eliminates the risk of contamination of food and other products (and ultimately humans) with E. coli O157. In other embodiments, the pharmaceutical composition is used as a prophylactic for preventing development of disease symptoms or lessening severity of symptoms in individiuals suspected of contracting E. coli O157 infection.
As discussed above, infection of humans with E. coli O157 may lead to diarrhea and serious complications that include hemorrhagic colitis, the hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and death. Specifically, the bacteria produce a toxin and other factors which causes damage to the lining of the intestine and blood vessels in other organs. The symptoms associated with E. coli O157 infection include severe cramping, diarrhea and vomiting. Thus, administering the pharmaceutical composition comprising at least one of bacteriophages V4, V5, V7, V8, V11 or V14 will reduce the level of E. coli O157 in the gastrointestinal tract of an individual in need of such a treatment by lysing the bacteria. This in turn will reduce the severity of symptoms associated with E. coli O157 infection in said individual.
In other embodiments, at least one of bacteriophages V4, V5, V5-re-isolated, V7, V8, V11 or V14 is administered to a matrix, for example, a water supply or a manure pile, to reduce the levels of E. coli O157 in said matrix. As will be appreciated by one knowledgeable in the art, reducing the levels of E. coli O157 in the matrix will in turn reduce the chance of subsequent contamination or infection.
The preferred delivery vehicles for the bacteriophages are in aqueous suspension, in a tablet or capsule form, as a powder or coating, or incorporated on or in material that can be eaten. However, as will be appreciated by one knowledgeable in the art, any suitable preparation which allows delivery of the bacteriophage into the gastrointestinal tract of the animal is within the scope of the invention. For examples of suitable carriers, diluents, excipients and the like, see Remington: The Science and Practice of Pharmacy, 2000, Gennaro, AR ed., Eaton, Pa. Mack Publishing Co.
In some embodiments, the pharmaceutical composition comprises bacteriophage at a titer of approximately 102-1010 PFU/ml or PFU/g. In other embodiments, the titer may be 105-109 PFU/ml or PFU/g. As will be apparent to one knowledgeable of the art, the titer used may vary according to, for example, the animal or individual being treated, the degree of infection and the state of disease progression.
In another aspect of the invention, there is provided a method of enriching for or selecting bacteriophage capable of lysing E. coli O157 under specific conditions. Specifically, a host carrying E. coli O157 in its gastrointestinal tract is fed a pharmaceutical composition as described above. In some embodiments, the pharmaceutical composition comprises at least one of V4, V5, V5-re-isolated, V7, V8, V11, V14 or preferably mixtures thereof. Gastrointestinal contents from the host are recovered over time and are filtered to recover bacteriophage. The filtrate is then plated onto host bacterial cells, for example, suitable strains of E. coli O157 and grown under conditions such that plaques form. As will be apparent to one knowledgeable of the art, the plaques contain the desired bacteriophage. Yet another aspect of the invention is directed to bacteriophage or mixtures of bacteriophages isolated by this method and the use thereof to reduce E. coli O157 levels in ruminant animals.